Since the tirst description of catalytic RNA s, or ribozymes, in 1982 , investigators recognized that the unique ability to catalytically cleave complementary target RNAs made these molecules potential therapeutic agents. Perhaps their most widespread use has been in investigations designed to inhibit replication of HIV- I, the causative agent of AIDS. Another new area of research at this time was the development of retroviral vectors as gene therapy delivery vehicles. The two lines of research seemingly made an excellent marriage and investigators began to generate retroviruses to deliver anti- HIV ribozymes to target cells. While some of these investigations showed initial promise, it has been difficult to draw conclusions based on data representing the use of various vectors delivering a multitude of expression cassettes and targeting a wide range of potential HIV-1 ribozyme cleavage sites. In addition, many studies failed to investigate ribozyme catalytic properties. It has also become obvious that the transfer of gene therapy techniques from in vitro to in vivo applications must meet several important requirements. These include methods to generate high retroviral titers for transduction of large numbers of target cells; methods to enrich gene-modified cells; and methods to obtain measurable long-term expression of therapeutic genes. For successful HIV gene therapy, the target cells of choice are hematopoietic stem cells (HSCs). Current strategies for targeting these cells require isolation, ex vivo manipulation including enrichment, followed by re-implantation into the patient. It is reasonable to assume that in vivo methods of enrichment would be superior to current methods of ex vivo enrichment, thereby circumventing long-term manipulation of HSCs resulting in reduced potential for contamination and phenotypic' alterations. Moreover, the most common method of ex vivo enrichment involves the use of the neomycin analog G418, which has been shown to decrease cell viability resulting in a reduced ability to repopulate the hematopoietic system. In this regard, mutant dihydrofolate reductase (DHFR) has been the focus of intense investigations to protect genetically modified HSCs from the myelosuppressive effect of in vivo antifolate chemotherapy, and therefore provides several advantages over other drug resistance markers commonly used in ribozyme studies and requiring ex vivo selection. The overall goals of this proposal are (1) to systematically investigate the catalytic properties of hammerhead ribozymes targeted to sites within the HIV- 1 genome, beginning with HIV- 1 tat sequences already studied by this investigator, and (2) to investigate the use of retroviruses expressing DHFWribozyme transgenes as potential gene therapy reagents for use in more complex systems.